Bone Marrow Proliferation Research Articles

Contribution of different bone marrow-derived cell types in endometrial regeneration using an irradiated murine model

To study the involvement of seven types of bone marrow-derived cells (BMDCs) in the endometrial regeneration in mice after total body irradiation. Prospective experimental animal study. University research laboratories. β-Actin-green fluorescent protein (GFP) transgenic C57BL/6-Tg (CAG-EGFP) and C57BL/6J female mice. The BMDCs were isolated from CAG-EGFP mice: unfractionated bone marrow cells, hematopoietic progenitor cells, endothelial progenitor cells (EPCs), and mesenchymal stem cells (MSCs). In addition three murine GFP(+) cell lines were used: mouse Oct4 negative BMDC multipotent adult progenitor cells (mOct4(-)BM-MAPCs), BMDC hypoblast-like stem cells (mOct4(+) BM-HypoSCs), and MSCs. All cell types were injected through the tail vein of 9 Gy-irradiated C57BL/6J female mice. Flow cytometry, cell culture, bone marrow transplantation assays, histologic evaluation, immunohistochemistry, proliferation, apoptosis, and statistical analysis. After 12weeks, histologic analysis revealed that uteri of mice with mOct4(-)BM-MAPCs and MSC line were significantly smaller than uteri of mice with uncultured BMDCs or mOct4(+) BM-HypoSCs. The percentage of engrafted GFP(+) cells ranged from 0.13%-4.78%. Expression of Ki-67 was lower in all uteri from BMDCs treated mice than in the control, whereas TUNEL(+) cells were increased in the EPCs and mOct4(+)BM-HypoSCs groups. Low number of some BMDCs can be found in regenerating endometrium, including stromal, endotelial, and epithelial compartments. Freshly isolated MSCs and EPCs together with mOct4(+) BM-HypoSCs induced the greatest degree of regeneration, whereas culture isolated MSCs and mOct4(-)BM-MAPCs transplantation may have an inhibitory effect on endometrial regeneration.

The function of a novel immunophenotype candidate molecule PD-1 in chronic lymphocytic leukemia

Programmed death-1 (PD-1) is a negative receptor expressed on lymphocytes including malignant B cells in chronic lymphocytic leukemia (CLL). In this work, we found that patients with CLL had a higher expression of PD-1 transcript (PDCD1) than healthy volunteers (p < 0.0001). PDCD1 expression was comparable between CLL cells from accumulation (peripheral blood) and proliferation (bone marrow) disease compartments. In blood samples of patients with mutated IGHV genes PDCD1 expression was higher than with unmutated IGHV (p = 0.0299). We demonstrated that phosphorylation of SYK and LYN, key B-cell receptor signaling kinases, was independent of PD-1 expression in patients with CLL, while ZAP-70 phosphorylation in negative tyrosine residue 292 showed strong inverse correlation (r = − 0.8, p = 0.0019). No associations between five single nucleotide polymorphisms of PDCD1, their expressions and susceptibility to CLL were found. In conclusion, PD-1 might be an independent, universal marker of CLL cells and a part of their activated phenotype, and subsequently might modulate the function of ZAP-70.

Erythroblast apoptosis and microenvironmental iron restriction trigger anemia in the VK*MYC model of multiple myeloma.

Multiple myeloma is a malignant disorder characterized by bone marrow proliferation of plasma cells and by overproduction of monoclonal immunoglobulin detectable in the sera (M-spike). Anemia is a common complication of multiple myeloma, but the underlying pathophysiological mechanisms have not been completely elucidated. We aimed to identify the different determinants of anemia using the Vk*MYC mouse, which spontaneously develops an indolent bone marrow localized disease with aging. Affected Vk*MYC mice develop a mild normochromic normocytic anemia. We excluded the possibility that anemia results from defective erythropoietin production, inflammation or increased hepcidin expression. Mature erythroid precursors are reduced in Vk*MYC bone marrow compared with wild-type. Malignant plasma cells express the apoptogenic receptor Fas ligand and, accordingly, active caspase 8 is detected in maturing erythroblasts. Systemic iron homeostasis is not compromised in Vk*MYC animals, but high expression of the iron importer CD71 by bone marrow plasma cells and iron accumulation in bone marrow macrophages suggest that iron competition takes place in the local multiple myeloma microenvironment, which might contribute to anemia. In conclusion, the mild anemia of the Vk*MYC model is mainly related to the local effect of the bone marrow malignant clone in the absence of an overt inflammatory status. We suggest that this reproduces the initial events triggering anemia in patients.

Dexamethasone enhances osteogenic differentiation of bone marrow- and muscle-derived stromal cells and augments ectopic bone formation induced by bone morphogenetic protein-2.

We evaluated whether dexamethasone augments the osteogenic capability of bone marrow-derived stromal cells (BMSCs) and muscle tissue-derived stromal cells (MuSCs), both of which are thought to contribute to ectopic bone formation induced by bone morphogenetic protein-2 (BMP-2), and determined the underlying mechanisms. Rat BMSCs and MuSCs were cultured in growth media with or without 10-7 M dexamethasone and then differentiated under osteogenic conditions with dexamethasone and BMP-2. The effects of dexamethasone on cell proliferation and osteogenic differentiation, and also on ectopic bone formation induced by BMP-2, were analyzed. Dexamethasone affected not only the proliferation rate but also the subpopulation composition of BMSCs and MuSCs, and subsequently augmented their osteogenic capacity during osteogenic differentiation. During osteogenic induction by BMP-2, dexamethasone also markedly affected cell proliferation in both BMSCs and MuSCs. In an in vivo ectopic bone formation model, bone formation in muscle-implanted scaffolds containing dexamethasone and BMP-2 was more than two fold higher than that in scaffolds containing BMP-2 alone. Our results suggest that dexamethasone potently enhances the osteogenic capability of BMP-2 and may thus decrease the quantity of BMP-2 required for clinical application, thereby reducing the complications caused by excessive doses of BMP-2. Highlights: 1. Dexamethasone induced selective proliferation of bone marrow- and muscle-derived cells with higher differentiation potential. 2. Dexamethasone enhanced the osteogenic capability of bone marrow- and muscle-derived cells by altering the subpopulation composition. 3. Dexamethasone augmented ectopic bone formation induced by bone morphogenetic protein-2.

Effect of water extract and distillate from the mixture of black goat meat and medicinal herb on osteoblast proliferation and osteoclast formation

Purpose: The effects of water extract and distillate from the mixture of black goat meat and medicinal herb on MG-63 osteoblast proliferation and mouse bone marrow derived osteoclast formation were investigated. Methods: Proximate composition, volatile basic nitrogen (VBN), mineral content, free amino acid composition and free fatty acid composition in black goat meat were determined. Water extract and distillate were prepared with three groups; goat meat only (BG-E, BGD), six herbs added group (BG-E6, BG-D6), and eight herbs added group (BG-E8, BG-D8). Osteoblast proliferation, mineralization and calcium uptake activity of MG-63 cells were measured and tartrate resistant acid phosphatase activity of osteoclasts was analyzed. Results: Black goat meat had remarkably low fat and high level of calcium. Glutamic acid was the most abundant amino acid. Herbs added extract groups (BG-E6 and BG-E8) showed increased MG-63 cell proliferation in a concentration dependent manner, while all the distillates did not show the effect. All extracts and distillates showed significantly increased osteoblast mineralization depending on the concentration. In particular, herb added extract, BG-E6, increased 170.3% of control and the distillate of BG-D and BG-D6 increased up to 168.5% and 159.8%, respectively. Calcium uptake activities of all water extracts showed remarkable increase of BG-E6 and BG-E8 up to 615.5% and 628.1% of control, respectively. Ditillates had no effect except BG-D6. All water extracts significantly reduced the activity of tartrate-resistant acid phosphatase (TRAP) in osteoclasts derived from mouse bone marrow. Conclusion: Combination of black goat meat and medicinal herb increased the MG-63 cell proliferation and effectively inhibited osteoclast differentiation in both water extracts and distillate of them, which implies that they could be used as potent functional food materials for bone health.

Recovery of the Normal B-Cell Compartment in Children Treated for B-Cell Precursor Acute Lymphoblastic Leukemia

BACKGROUNDCytotoxic treatment in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients induces a dramatic decrease in B-cell precursor (BCP) and mature B-cell numbers, followed by regeneration of BCPs in the bone marrow (BM) and subsequent replenishment of mature B-cells in the peripheral blood (PB) in between treatment blocks and after stop of treatment. To understand the degree of B-cell recovery after such dramatic changes, we first evaluated the composition of the B-cell population in the BM and PB of pediatric BCP-ALL patients during and after therapy. Secondly, we investigated whether the immunophenotypic maturation of BCPs in regenerating BM is similar to normal BCP development or whether such regeneration induces immunophenotypic aberrancies, which could potentially hamper minimal residual disease (MRD) detection. Finally, we assessed whether compensatory proliferation plays a role during B-cell regeneration, since enhanced proliferation might limit the B-cell receptor diversity and consequently may affect susceptibility to infections during and after therapy.METHODSFor immunophenotypic characterization of different B-cell subsets, 8-color flow cytometry was performed on fresh BM and PB samples at different time points after start of therapy (DCOG ALL11-protocol). To study BCP maturation, a multidimensional maturation pathway based on 5 backbone markers was designed and the expression pattern of several differentiation markers during this maturation pathway was evaluated. To assess proliferation in BCP subsets, BM samples were stained with subset-specific antibodies and DRAQ5 for cell cycle analysis. The proliferation history of sorted pre-B-II-small and immature subsets in BM and sorted mature B-cell subsets in PB was assessed by performing a kappa-deleting recombination excision circle (KREC)-assay.RESULTSBCP regeneration occurred mainly at day 78, month 5 and after stop of therapy. The BCP compartment in regenerating BM at time points during therapy showed a shift towards the most immature stages. In PB, mature B-cell numbers decreased after start of therapy and newly generated mature B-cells subsets reappeared at month 5 and after stop of therapy.Importantly, the BCP maturation pathway with its expression patterns of CD10, CD34, CD58, CD66c, CD38, CD123, CD9, CD81, CD24, TdT, Igκ and Igλ was comparable between regenerating BM and BM of healthy individuals, albeit that a shift in the relative BCP subset distribution was observed in regenerating BM.As expected, most proliferation in BM of healthy controls occurred in the pre-B-II-large subset (68% ±11% (mean ±SD) proliferating cells). Comparable percentages of proliferating pre-B-II-large cells were found in regenerating BM: 74%±10% at day 78, 72%±10% at month 5 and 63% (preliminary data, n=1) at one year after stop of therapy (month 36). Also pre-B-I cells showed some proliferation, with no significant differences between normal and regenerating BM (Figure 1). If present, the pre-B-II-small and immature BCP subsets showed no proliferation in regenerating and normal BM. KREC-analysis of sorted pre-B-II-small and immature subsets confirmed that no cell divisions had occurred after IGK-rearrangement in normal BM as well as regenerating BM at month 5 and month 36. Low numbers of pre-B-II-small and immature cells precluded KREC-analysis at day 78.KREC-analysis of the various mature B-cell subsets in PB showed no significant difference in proliferation history between PB of patients at different time points during or after therapy and PB of healthy controls.CONCLUSIONSIn BCP-ALL patients, the B-cell compartment is drastically affected during treatment. Subsequent regeneration of BCPs and mature B-cells occurs at different time points during therapy and after stop of therapy. Immunophenotypically, BCP maturation in regenerating BM is similar to normal B-cell differentiation, indicating that MRD detection will not be hampered by aberrant immunophenotypes of regenerating BCPs. Importantly, no enhanced proliferation is observed in BCP subsets in BM and mature B-cells subsets in PB of patients during and after therapy. The lack of compensatory proliferation suggests that B-cell regeneration is due to a larger influx of non-committed stem cells into the B-cell lineage and indicates that a diverse immune repertoire will most likely be restored during recovery of the B-cell compartment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 17399: Glutathione-S-Transferase P Regulates Neovascularization in the Remodeling Heart via Effects on Endothelial Progenitor Cells and Reparative Monocytes

Glutathione- S -transferase P (GSTP) modulates c-Jun N-terminal kinase (JNK) activation and the proliferation of bone marrow (BM) myeloid progenitors; however, its effects on endothelial progenitor cells (EPCs) and neovascularization are unknown. Hypothesis: GSTP promotes EPC function in a JNK-dependent manner, thereby supporting neovascularization in heart failure (HF). GSTP-/- mice exhibited significantly (p&lt;0.01 vs. wild type [WT]) reduced circulating and BM EPCs, increased pro-inflammatory F4/80 + CD11b + Gr-1 hi monocytes, diminished BM EPC function (proliferation, migration, and vascular tube formation), and increased BM JNK phosphorylation. These effects were reversed upon treatment with the JNK inhibitor SP600125. HF was induced by left coronary ligation in WT and GSTP-/- mice (sham control). GSTP-/- HF mice exhibited (p&lt;0.05 vs. WT HF): 1) fewer circulating and BM EPCs and increased pro-inflammatory monocytes; 2) exaggerated left ventricular (LV) remodeling and inflammation; and 3) reduced myocardial capillary density. Next, chimeric mice were generated via BM ablation in WT mice and reconstitution with either GSTP-/- (GSTP-/-c) or WT (WTc) BM. GSTP-/-c and WTc mice underwent coronary ligation or sham operation (n = 8-15/group). As compared with WTc HF, GSTP-/-c HF hearts had significant (p &lt; 0.01): 1) exaggeration of LV remodeling and dysfunction; 2) increases in remote zone fibrosis; 3) reductions in capillary density and VEGF-A, -B, and -C gene expression; and 4) augmentation of interleukin (IL)-1β and IL-6 expression. GSTP-/-c mice also exhibited (p &lt; 0.01 vs. WTc mice) increased BM JNK activation, reduced circulating EPCs, increased pro-inflammatory CD11b + Ly6C hi monocytes, but comparable Ly6C low monocytes at baseline. Moreover, at 3 and 7 d post-ligation, GSTP-/-c HF mice had fewer circulating EPCs and reparative Ly6C low monocytes, but significantly higher Ly6C hi monocytes. Conclusion: GSTP promotes EPC function and reparative monocyte mobilization, and suppresses inflammation, thereby improving angiogenic gene expression, neovascularization and LV remodeling after infarction. Hence, human GSTP polymorphisms, which are frequent, may impact cardiac reparative capacity in patients with myocardial infarction.

ChemInform Abstract: Amphirionin‐5, a Novel Linear Polyketide from a Cultured Marine Dinoflagellate Amphidinium Species with a Potent Cell Proliferation‐Promoting Activity.

Abstract A novel linear polyketide, amphirionin-5 (1), has been isolated from the cultivated algal cells of the benthic dinoflagellate Amphidinium sp. (strain KCA09053), and the structure was elucidated on the basis of detailed analyses of 1D and 2D NMR data. Amphirionin-5 (1) resulted in a 282% increase in the proliferation of murine bone-marrow derived stromal ST-2 cells and 320% increase in the proliferation of murine osteoblastic MC3T3-E1 cells.

Lithium stimulates human bone marrow derived mesenchymal stem cell proliferation through GSK-3β-dependent β-catenin/Wnt pathway activation.

Mesenchymal stem cells (MSCs) are multipotent cells that have been widely used in cell based transplantation therapy. The use of MSCs requires invitro expansion in order to fulfill their regenerative capacity. Therefore the proliferative ability of MSCs is one of the key factors which determine MSC therapeutic efficacy. In the present study, we showed for the first time that lithium, a well-known antidepressant, reversibly promoted the proliferation of human bone marrow derived MSCs invitro. MSCs treated with 5mm lithium proliferated more rapidly than untreated cells without undergoing apoptosis. Lithium increased the proportion of cells in S phase as well as cyclin D1 expression. Mechanistic studies revealed that these effects were dependent upon the activation of the glycogen synthase kinase 3β (GSK-3β) mediated canonical Wnt pathway. Lithium induced Ser9 phosphorylation, which results in the inhibition of GSK-3β activity, β-catenin accumulation and Wnt pathway activation. Utilizing a specific GSK-3β inhibitor SB216763 or siRNA-mediated inhibition of GSK-3β produced effects similar to those induced by lithium. In contrast, either quercetin, an inhibitor of the β-catenin/TCF pathway, or siRNA-mediated knockdown of β-catenin abolished the proliferative effect of lithium, suggesting that lithium stimulates MSC proliferation via the GSK-3β-dependent β-catenin/Wnt pathway. Collectively, these studies elucidate a novel role of lithium, which may not only provide a simple and effective way to strengthen MSC transplantation therapy efficacy but also shed light on lithium's clinical application for the treatment of certain disorders resulting from β-catenin/Wnt pathway suppression.

Translational initiation regulated by ATM in dendritic cells development

Ataxia telangiectasia mutated (ATM) protein has been implicated in multiple pathways such as DNA repair, cell cycle checkpoint, cell growth, development, and stem cell renewal. In this study, we demonstrate evidence that ATM is involved in granulocyte macrophage colony-stimulating factor (GM-CSF)-induced dendritic cell (DC) development from bone marrow (BM) cells. Inactivation of ATM protein results in decreased BM proliferation, leading to reduced DC development and their activity for T cell activation. Expression of Jak2, STAT5, and mTOR is suppressed in both wild-type and ATM-null BM prior to GM-CSF stimulation. Activation of those proteins is delayed and prolonged hypophosphorylation of 4EBP1 is observed in ATM-null BM when treated with GM-CSF, although Erk and p38 are similarly expressed and activated in both wild-type and ATM-null BM cell types. Akt is also suppressed in wild-type BM, and transduction of constitutively active Akt or STAT5 in ATM-null BM restores DC development. Together, these results illustrate that ATM deficiency causes impaired initiation of protein translation in BM, leading to immature development of DC.

Monitoring residual disease in the ph-negative myeloproliferative neoplasms post-allogeneic stem cell transplantation: more mutations and more methodologies.

Monitoring residual disease in the ph-negative myeloproliferative neoplasms post-allogeneic stem cell transplantation: more mutations and more methodologies.

Biological effects of sol-gel derived ZrO2 and SiO2/ZrO2 coatings on stainless steel surface--In vitro model using mesenchymal stem cells.

The objective of this study was to determine biocompatibility of zirconia-based coatings obtained by the sol-gel method. Two matrices, ZrO2 and SiO2/ZrO2, were created and applied on stainless steel type 316L with dip-coating technique. The morphology and topography of biomaterials' surface were characterized using energy-dispersive X-ray spectroscopy and atomic force microscopy, while chemical composition was analyzed by Raman spectroscopy. Additionally, wettability and surface free energy were characterized. Biocompatibility of obtained biomaterials was evaluated using an invitro model employing mesenchymal stem cells (MSCs) of adipose and bone marrow origin. Biological analysis included determination of proliferation activity and morphology of MSCs in cultures on synthesized biomaterials. Osteoinductive properties of biomaterials were determined both in non-osteogenic, as well as osteogenic conditions. The results showed that investigated biomaterials exerted different impact on MSCs. Biomaterial with ZrO2 layer was more biocompatible for adipose-derived MSCs, while SiO2/ZrO2 layer promoted proliferation of bone marrow derived MSCs. Moreover, hybrid coating exhibited greater osteoinductive properties than ZrO2 coating, both on cultures with adipose-derived stromal (stem) cells and bone marrow stromal cells. Observed biological effects may result not only from different chemical composition, but also from diverse wettability. The ZrO2 coating was characterized as hydrophobic layer, while SiO2/ZrO2 exhibited hydrophilic properties. The results obtained suggest that behavior of MSCs in response to the biomaterial may vary depending on their origin, therefore we postulate, that screening analysis of implants' biocompatibility, should incorporate model applying both adipose- and bone marrow derived MSCs.

Abstract 347: Glutathione-S-Transferase P Regulates Bone Marrow Derived Endothelial Progenitor Cell (EPC) Function and Neovascularization in the Infarcted Heart

Glutathione-S-transferase P (GSTP) modulates proliferation of bone marrow (BM) myeloid progenitors via non-catalytic inhibition of c-Jun N-terminal kinase (JNK); however, its effects on BM-derived EPCs are unknown. We hypothesized that GSTP supports EPC mobilization while suppressing inflammatory cells, thereby promoting tissue neovascularization. We generated chimeric mice using BM-ablated wild-type (WT) mice reconstituted with either GSTP-/- (GSTP-/-c) or WT (WTc) BM. GSTP-/-c and WTc mice underwent coronary ligation or sham operation (n = 8-15/group). Compared to WTc sham, 4 w after surgery, WTc HF mice exhibited significant (p &lt; 0.05): LV dilatation, dysfunction, and fibrosis; increased mortality; and augmented circulating pro-inflammatory CD11b+Ly6Chi monocytes, but comparable circulating CD34+VEGFR2+ EPCs. In contrast, compared to WTc HF, GSTP-/-c HF hearts had significant (p &lt; 0.01): 1) worsening of LV dilatation (LVEDV 118 ± 30 vs 88 ± 13 μL), dysfunction (LVEF 21 ± 4 vs 32 ± 9 %), wall thinning (anterior wall thickness 0.41 ± 0.06 vs 0.49 ± 0.1 mm), and LV hypertrophy (LV/tibia length 4.9 ± 0.5 vs 4.5 ± 0.6 mg/mm); 2) increased remote zone fibrosis by trichrome staining (1.3 ± 0.2 vs 0.61 ± 0.3 %); 3) reduced capillary density (476 ± 60 vs 544 ± 37 capillary/mm2) and increased capillary area (14 ± 1.1 vs 12 ± 1.2 μm2); 4) diminished gene expression of VEGF A, VEGF B, and VEGF C; 5) and increased IL-1β, IL-6 gene expression. GSTP-/-c mice also exhibited (p &lt; 0.01) increased BM JNK activation, reduced circulating EPCs (0.18±0.07 vs 0.24±0.06%), increased CD11b+Ly6Chi monocytes (2.25±0.5 vs 1.8±0.6%), but comparable anti-inflammatory CD11b+Ly6Clow monocytes at baseline as compared with WTc mice. Moreover, at 3 and 7 d after ligation, GSTP-/-c HF mice exhibited fewer circulating EPCs and anti-inflammatory monocytes, and significantly higher pro-inflammatory monocytes as compared with WTc HF mice. We conclude that GSTP promotes EPC and reparative monocyte mobilization, thereby improving angiogenic gene expression, neovascularization and LV remodeling after myocardial infarction. This suggests that GSTP polymorphisms can impact cardiac reparative capacity in humans, and that enhancing GSTP function after infarction may yield therapeutic benefit.

Stressing out stem cells: linking stress and hematopoiesis in cardiovascular disease.

Stress has long been thought to be a major contributing factor to cardiovascular disease, although little is known about the underlying cellular mechanisms. A new study in humans and mice suggests that chronic stress promotes hematopoietic stem cell proliferation in bone marrow, leading to increased leukocyte production, circulation and recruitment to the vasculature ( pages 754–758 ).

Platelet factor 4 protects bone marrow mesenchymal stem cells from acute radiation injury.

The aim of this study was to find a new radiation protector, platelet factor 4 (PF4) and to identify its effect on haemopoietic microenvironment in vitro and in vivo. Radiation damage on bone marrow mesenchymal stem cells ex and in vitro was set up as models. Growth curve analysis, clonogenic survival assay, FACSCalibur™ (BD Immunocytometry Systems, San Jose, CA), 5-ethynyl-2'-deoxyuridine immunofluorescence staining and quantitative reverse transcription-polymerase chain reaction were employed to assess the characterization of bone marrow mesenchymal stem cells (BMSCs), proliferation, apoptosis, cell cycle and gene expression. A dose- and time-dependent enhancement of cell viability and survival was observed for PF4 treatment along with 500 cGy γ-radiation in vitro. The same phenomena were noted in vivo, including enhancement of adherence and proliferation ability while inhibition of cell apoptosis, which were associated with a short-term decrease in the G0/G1 ratio owing to S phase arrest. These were accompanied with enhanced Bcl-2 expression and p53/p21 loss. These results uncover that PF4 might be a novel therapeutic approach, which could reduce DNA damage and increase survival of BMSCs, in part, by inhibiting p53/p21 axis and facilitating DNA damage repair. This study explores the feasibility of a new radioprotector and hence may be clinically important.

Dose-response of superparamagnetic iron oxide labeling on mesenchymal stem cells chondrogenic differentiation: a multi-scale in vitro study.

AimThe aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues.MethodsMSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity.ResultsNo influence of various SPIO concentrations was noted on human bone marow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-β1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations.ConclusionsThis study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-β1 driven chondrogenesis in collagen sponges. Low concentrations (12.5–25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.

TU-F-12A-02: Quantitative Characterization of Normal Bone Marrow Proliferative Activity with FLT PET/CT

Purpose: [F-18]FLT PET is a tool for assessing health of bone marrow by evaluating its proliferative activity. This study establishes a baseline quantitative characterization of healthy marrow proliferation to aid in diagnosis of hematological disease. Methods: 31 patients (20 male, 11 female, 41–76 years) being treated for solid cancers with no history of hematological disease, osseous metastatic disease, or radiation therapy received pre-treatment FLT PET/CT scans. Total bone marrow was isolated from whole body FLT PET images by manually removing organs and applying a standardize uptake value (SUV) threshold of 1.0. Because adult marrow is concentrated in the axial skeleton, quantitative total bone marrow analysis (QTBMA) was used to isolate marrow in the lumbar spine, thoracic spine, sacrum, and pelvis for analysis. SUV mean , SUV max , and SUV CV were used to quantify bone marrow proliferation. Correlations were explored between SUV and patient characteristics including age, weight, height, and BMI using the Spearman coefficient (ρ). Results: The population-averaged whole-skeleton SUV mean , SUV max , and SUV CV were 3.0±0.6, 18.4±5.7, and 0.6±0.1, respectively. Uptake values in the axial skeleton were similar to the whole-skeleton demonstrated by SUV mean in the thoracic spine (3.6±0.6), lumbar spine (3.3±0.5), sacrum (3.0±0.6), and pelvis regions (2.8±0.5). Whole-skeleton SUV max correlated with patient weight (ρ=0.47, p<0.01) and BMI (ρ=0.60, p<0.01), suggesting marrow activity is related to the body's burden. SUV measures in the thoracic spine, lumbar spine, sacrum, and pelvis were negatively correlated with age (ρ:−0.41 to −0.46, p≤0.02). These negative correlations reflect the fact that active marrow in the adult skeleton is localized in the axial skeleton and decreases with age. Conclusions: Normal bone marrow characterizations were determined using FLT PET. These results provide a baseline characterization against which proliferative activity of abnormal marrow can be compared.

In vitro and in vivo biocompatibility of multi-walled carbon nanotube/biodegradable polymer nanocomposite for bone defects repair

Biomaterials are extensively used in bone defect recovery caused by bone diseases. Multi-walled carbon nanotubes have been reported to reinforce synthetic polymeric materials. The aim of the study is to test poly(3-hydroxybutyrate- co-3-hydroxyvalerate) loaded with different amounts of multi-walled carbon nanotubes to fabricate nanocomposites. Mechanical, mineralization, and degradation properties were studied in vitro. The proliferation and differentiation of rat bone marrow stem cells were studied to determine biocompatibility in vivo. The incorporation of multi-walled carbon nanotubes greatly increased the mechanical properties of poly(3-hydroxybutyrate- co-3-hydroxyvalerate) and the strongest composite obtained was at 2% multi-walled carbon nanotubes. The 2% nanocomposite also had higher rat bone marrow stem cell adhesion, proliferation, and differentiation characteristics compared to the pure poly(3-hydroxybutyrate- co-3-hydroxyvalerate). The apoptosis in the later stage of rat bone marrow stem cells decreased in the 2% nanocomposites group at different time points. Based on histology and micro-computed tomography tests 6 weeks after in vivo implantation, the 2% multi-walled carbon nanotubes/poly(3-hydroxybutyrate- co-3-hydroxyvalerate) treated animals had a higher volume of bone formation compared to the pure poly(3-hydroxybutyrate- co-3-hydroxyvalerate) group. Thus, the presence of multi-walled carbon nanotubes has an apparent positive effect on poly(3-hydroxybutyrate- co-3-hydroxyvalerate) in assisting osteogenesis.

Amphirionin-5, a novel linear polyketide from a cultured marine dinoflagellate Amphidinium species with a potent cell proliferation-promoting activity

A novel linear polyketide, amphirionin-5 (1), has been isolated from the cultivated algal cells of the benthic dinoflagellate Amphidinium sp. (strain KCA09053), and the structure was elucidated on the basis of detailed analyses of 1D and 2D NMR data. Amphirionin-5 (1) resulted in a 282% increase in the proliferation of murine bone-marrow derived stromal ST-2 cells and 320% increase in the proliferation of murine osteoblastic MC3T3-E1 cells.

Development of Gelatin-Thai Silk Fibroin Microspheres for Three Dimensional Cell Culture

Microspheres have been widely used for tissue engineering scaffolds. Microspheres have many advantages over the macrostructure such as high surface area for cell adhesion and proliferation and low mass transfer limits. In this study, we fabricated microspheres from gelatin and silk fibroin using water-in-oil (w/o) emulsion technique and glutaraldehyde crosslinking. Gelatin (G) and silk fibroin (SF) were blended at different G/SF weight blending ratios of 100/0, 90/10, 70/30, and 50/50. Physical and chemical properties of the microspheres including size and morphology were characterized. The Average size of microspheres obtained were at 858.42±41.93, 832.97± 9.44 , 785.24±17.66 and 735.83 ±13.19 μm, respectively. Morphology of G/SF microspheres was observed under a scanning electron microscope. Blending of silk fibroin increased the crosslinking degrees and water absorption. It also reduced degradation rate, comparing to the gelatin microspheres. Thein vitroattachment and proliferation of rat bone marrow derived mesenchymal stem cells (MSC) cultured on G/SF microspheres were evaluated. G/SF 50/50 microspheres promoted the highest attachment of MSC on microspheres (46.0±5.8% of initial seeding at 6 hr). The G/SF 70/30 microspheres promoted the higher cell proliferation of MSC compared the others. Specific growth rate of the cells on the microspheres was at 9.85x10-3h-1.